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F J Schmidt

Publications and source records attributed to F J Schmidt.

At least 19 recordsLinked to original sources
PubMed1997

Interacting RNA species identified by combinatorial selection.

RNA molecules were selected from a random sequence library for their ability to bind to an RNA stem-loop target. Oligonucleotides with extensive Watson-Crick complementarity to the RNA ligand were selected against by inclusion of a blocking oligodeoxynucleotide in the binding phase of the selection protocol. After 18 generations of SELEX (systematic evolution of ligands by exponential enrichment) a single RNA family was predominant in the binding population. The winning aptamer RNA bound the target RNA with an apparent Kd = 70 nM. Structural mapping and Fe(II)-EDTA protection indicated that the target RNA interacted with small unpaired loops in the aptamer structure.

Base Composition↗
PubMed1996

RNA libraries and RNA recognition.

Random RNA libraries, consisting of > 10(13) unique sequences, contain molecules capable of specifically binding small molecule and protein ligands by noncovalent interaction. Successive steps of affinity purification and amplification allow the propagation and eventual isolation of specific binding molecules, called aptamers. Although protein- and small-molecule-binding aptamers have been characterized previously, selection of RNA aptamers capable of binding nucleic acids has previously yielded only molecules capable of Watson-Crick base pairing to the nucleic acid ligand used for selection. On the other hand, it is known from studies on catalytic and other RNAs that both inter- and intramolecular RNA-RNA interaction can occur by non-Watson-Crick means. We have therefore incorporated a strategy to obviate the possibility of Watson-Crick interaction into a selection scheme for the isolation of RNA-recognizing aptamers. The aptamers so isolated do not show extensive Watson-Crick complementarity with the RNA ligand used for the selection, thereby validating the selection strategy. Curiously, all of the aptamers characterized contain several oligo-G stretches bounded by U residues. This sequence motif, which occurs as DNA in telomeres (chromosome ends) may therefore be a general RNA-RNA interaction motif. An additional sequence motif is apparently superimposed on this background structure.

Bacillus subtilis↗
PubMed1994

Gene organization in the bleomycin-resistance region of the producer organism Streptomyces verticillus.

A nucleotide sequence of 7 kb is reported, encompassing two bleomycin-resistance (BmR-encoding) genes and five other open reading frames (ORFs) from the Bm-producing organism Streptomyces verticillus ATCC 15003. The deduced ORFs, in sequence order, encode for (i) a protein homologous to an amino-acid dioxygenase; (ii) BlmA, the BmR-binding protein described by Sugiyama et al. [Gene 151 (1994) 11-16]; (iii) a product containing three copies of a sequence homologous to the ankyrin repeat; (iv) a product lacking homology to any of the sequences in the Protein Identification Resource database (PIR), release 37; (v) BlmB, the BmR acetyltransferase described by Sugiyama et al. (1994); (vi) an unidentified protein which augmented resistance determined by ORF2 (BlmA); (vii) a member of the ATP-binding cassette (ABC) family of transport protein. Predicted translational frameshifts in the -1 frame occur at the junctions between ORF3 and ORF4, ORF4 and ORF5, and ORF6 and ORF7. Sequences homologous to ORF2 and ORF3 were identified in the genome of the producer organism for the related antibiotic phleomycin.

Acetyltransferases↗
PubMed1994

Molecular cloning of sheep lung dipeptidase: a glycosyl phosphatidylinositol-anchored ectoenzyme that converts leukotriene D4 to leukotriene E4.

Sheep lung dipeptidase was released from a plasma membrane preparation by digestion with a phosphatidylinositol-specific phospholipase C. The dipeptidase was purified to homogeneity using affinity chromatography followed by high performance liquid chromatography. The NH2-terminal sequence was determined and employed to prepare a probe to clone the c-DNA of the enzyme. The primary structure of sheep lung dipeptidase, deduced from the c-DNA exhibited a high homology to kidney dipeptidases cloned from other animal species. Northern analysis detected the m-RNA of the dipeptidase in lung, kidney, and intestinal tissues of the sheep.

Amino Acid Sequence↗
PubMed1993

Suppression of loss-of-function mutations in Escherichia coli ribonuclease P RNA (M1 RNA) by a specific base-pair disruption.

A plasmid encoding ribonuclease P RNA of Escherichia coli (M1 RNA) was mutagenized with hydroxylamine in vitro and defective rnpB genes were identified by screening in an in vivo suppression assay. Defective rnpB sequences were mutagenized with a second round of hydroxylamine to restore activity. We report here that conversion of the C32.G48 base-pair of RNase P RNA to either C.A or U.G restored activity to defective rnpB genes bearing a variety of spatially distinct primary mutations. Disruption of this base-pair in an otherwise wild-type rnpB sequence increased the growth rate of the indicator strain E. coli FS101, consistent with the opening of C32.G48 during in vivo assembly of or catalysis by RNase P.

Base Sequence↗
PubMed1993

Glucose concentration in subcutaneous extracellular space.

OBJECTIVE: To compare the subcutaneous glucose sensor measurements with two reference methods. Previous studies provide conflicting findings about the real glucose concentrations in subcutaneous tissue. Some suggest substantially lower concentration, whereas others measure proportionally higher glucose concentrations compared with the blood compartment. Before these results can be taken seriously as an expression of the real glucose concentration in the extracellular space, the measurements must be validated by an independent method. RESEARCH DESIGN AND METHODS: We applied a microdialysis-based enzyme sensor to measure glucose concentration in subcutaneous tissue. We also developed two reference methods: subcutaneous filtrate collection and an equilibration method using ultrafiltration membranes to support the earlier findings. We provided an anatomical model to explain the results. RESULTS: The mean overall intercellular filtrate glucose concentration, sampled with the filtrate collector and taken after a 6-h stabilization time, including the values during the glucose clamp period, was 46 +/- 9%. The mean subcutaneous glucose concentration measured with the glucose sensor, calibrated in vitro, was 44 +/- 8% of the mean venous blood glucose concentration. Mean overall intercellular equilibrate glucose concentration, i.e., the mean glucose concentration in the subcutaneous extracellular space, taken after a 4-h stabilization time, was 46 +/- 15% of the mean venous blood glucose concentration. CONCLUSIONS: The close agreement between the mean values of subcutaneous glucose concentrations, obtained with three independent methods--filtration, equilibration, and dialysis (sensor)--shows the real glucose concentration in subcutaneous interstitial fluid is approximately 50% the blood glucose value in normal humans. Our results clarify some of the conflicting evidence presented in previous studies.

Adult↗
PubMed1992

Sequences encoding the protein and RNA components of ribonuclease P from Streptomyces bikiniensis var. zorbonensis.

The genes encoding the RNA (rnpB) and protein (rnpA) subunits of ribonuclease P (RNase P) of Streptomyces bikiniensis var. zorbonensis have been cloned by complementing the temperature-sensitive growth phenotype of Escherichia coli strains that carry mutations in these genes. The rnpB sequence of S. bikiniensis includes new covariations that lead to refinement of the previous secondary structure models for RNase P RNAs. The deduced amino acid sequence of S. bikiniensis RNase P is conserved with that of other known RNase P proteins only to a limited extent. Immediately upstream from rnpA is an open reading frame that codes for the highly conserved ribosomal protein, L34. This same gene arrangement occurs in all bacteria studied to date.

Amino Acid Sequence↗
PubMed1992

Conserved gene arrangement in the origin region of the Streptomyces coelicolor chromosome.

A 23-kb fragment of the Streptomyces coelicolor chromosome spanning the dnaA region has been isolated as a cosmid clone. Nucleotide sequence analysis of a 5-kb portion shows that the genes for the RNase P protein (rnpA), ribosomal protein L34 (rpmH), the replication initiator protein (dnaA), and the beta subunit of DNA polymerase III (dnaN) are present in the highly conserved gene arrangement found in all eubacterial genomes studied so far. The dnaA-dnaN intergenic region is approximately 1 kb and contains a cluster of at least 12 DnaA boxes with a consensus sequence of TTGTCCACA matching the consensus DnaA box in the phylogenetically related Micrococcus luteus. Two DnaA boxes precede the dnaA sequence. We propose that the chromosomal origin (oriC) of S. coelicolor lies between dnaA and dnaN. In related work, J. Zakrzewska-Czerwinska and H. Schrempf (J. Bacteriol. 174:2688-2693, 1992) have identified the homologous sequence from the closely-related Streptomyces lividans as capable of self-replication.

Amino Acid Sequence↗
PubMed1992

Calibration of a wearable glucose sensor.

Calibration of glucose sensors proved difficult for electrodes with immobilized glucose-oxidase. The correlation between the sensitivity of the electrodes in vitro and in vivo appeared to be poor. We developed a new type of glucose sensor, based on a microdialysis system, in which an oxygen electrode is used as detector outside the body and the enzyme glucose-oxidase dissolved in water is used as a dynamic selector. The enzyme solution is pumped through a hollow fiber placed subcutaneously, before the fluid passes the detector. The glucose sensor was tested in the subcutaneous abdominal tissue of 12 healthy volunteers and 12 type I diabetic patients. Blood glucose was clamped at two levels to permit a two-point calibration of the sensor in vivo. These values correlated well with the in vitro calibration factors (r = 0.949). In subcutaneous tissue the sensor measures 43 +/- 9% of the blood glucose value, using the in vitro calibration factor. No differences were detected between healthy volunteers and diabetic patients.

Adult↗
PubMed1991

Transcript hairpin structures are not required for RNA polymerase pausing in the gene encoding the E. coli RNase P RNA, M1 RNA.

Strong pauses at nucleotides +118 and +121 relative to the transcriptional start occur during in vitro transcription of the E. coli rnpB gene encoding the catalytic M1 RNA subunit of Ribonuclease P. These pauses are immediately downstream of 2 phylogenetically conserved stem-loop structures in the RNA. In the present work, single-base changes which disrupted Watson-Crick base-pairing in the hairpins were introduced into rnpB. Transcription studies in vitro with these modified templates revealed that none of the nucleotide changes predicted to increase or decrease the stability of the first hairpin significantly affected the pause half-lives. A mutation which disrupted the second hairpin increased the pause half-life 2-fold. The data suggest that the upstream stem and loop structures in the transcript are not involved in the pausing event.

Base Sequence↗
PubMed1991

Long-range structure in ribonuclease P RNA.

Phylogenetic-comparative and mutational analyses were used to elucidate the structure of the catalytically active RNA component of eubacterial ribonuclease P (RNase P). In addition to the refinement and extension of known structural elements, the analyses revealed a long-range interaction that results in a second pseudoknot in the RNA. This feature strongly constrains the three-dimensional structure of RNase P RNA near the active site. Some RNase P RNAs lack this structure but contain a unique, possibly compensating, structural domain. This suggests that different RNA structures located at different positions in the sequence may have equivalent architectural functions in RNase P RNA.

Bacillus subtilis↗
PubMed1991

Development of a wearable glucose sensor; studies in healthy volunteers and in diabetic patients.

A glucose sensor with a subcutaneous dialysis system was tested in six healthy volunteers during an oral glucose tolerance test and in ten diabetic patients with hyperglycemia during rapid decline of blood glucose levels. There was a good correlation between sensor and blood glucose values. During oral glucose tolerance tests in the volunteers, there was a mean delay of 4.4 minutes in the rise of the value registered subcutaneously and of 8.2 minutes in the fall of the curves. In the diabetic patients the maximum delay was 22 minutes. Nine days after insertion of the dialysis system it was still functioning well.

Adult↗
PubMed1990

Construction and processing of transfer RNA precursor models.

Several "dimeric" tRNA molecules were constructed as potential substrates for ribonuclease P (RNase P) and for M1 RNA, the catalytic subunit of RNase P. Construction was affected by the T4 RNA ligase-mediated coupling of a mature Escherichia coli tRNA (acceptor substrate) and nucleotides 1-36 of yeast tRNAPhe (donor substrate), followed by annealing of the 3'-half of yeast tRNAPhe (nucleotides 38-76). E. coli RNase P and M1 RNA were both found to cleave the dimeric tRNA precursor model constructed from E. coli tRNAPhe (5'-tRNA) and yeast tRNAPhe (3'-tRNA) in a reaction that was dependent on the presence of the annealed 3'-half molecule derived from yeast tRNAPhe, or on some conformation imposed by the presence of this species; the product had the same mobility as authentic E. coli tRNAPhe on a polyacrylamide gel. By utilizing tRNA precursor models radiolabeled at phosphodiesters immediately preceding or following the putative site of processing, cleavage of the substrate by both M1 RNA and the holoenzyme was demonstrated to occur at the expected phosphate ester linkage. The results obtained here suggest that the endonucleolytic separation of two tRNAs by RNase P is dependent on one or more structural features in the 3'-half of the 3'-tRNA, and thus are consistent with the report of McClain et al. (McClain, W. H., Guerrier-Takada, C., and Altman, S. (1987) Science 238, 527-530) that identifies the T stem and loop as a possible recognition site.

Base Sequence↗
PubMed1990

Development of a potentially wearable glucose sensor for patients with diabetes mellitus: design and in-vitro evaluation.

A potentially wearable glucose sensor was developed, consisting of an oxygen electrode as detector and a dynamic enzyme perfusion system as selector. The selector is a hollow fibre, which can be placed subcutaneously and dialyses glucose from tissue fluid. In this design the problems of enzyme instability and oxygen limitation might be circumvented. The sensor measures glucose reliably for over two weeks, provided a new 10 ml syringe containing a glucose oxidase solution is connected to the system each day.

Biosensing Techniques↗
PubMed1989

Sites of initiation and pausing in the Escherichia coli rnpB (M1 RNA) transcript.

DNA sequences affecting the transcription of the Escherichia coli rnpB transcript encoding the catalytic M1 RNA subunit of RNase P have been analyzed. Previous work (Motamedi, H., Lee, Y., and Schmidt, F.J.) (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3959-3963) identified S1 nuclease protection products corresponding to transcripts originating upstream of the M1 RNA gene. Sequence analysis of the upstream region of rnpB identified three regions homologous to the E. coli consensus promoter sequence. In the present work, analysis of in vitro transcription products by S1 nuclease mapping indicated that all three promoter homologies were capable of directing transcription. The nearest promoter, P-1, was approximately 100 times more active than either of the upstream homologies P-2 and vivo experiments, wherein the three promoter homologies preceding rnpB were cloned into the galactokinase (GalK) expression vector pKO100. The promoter homology nearest to the M1 RNA gene directed the synthesis of GalK above background. The upstream promoter homologies did not direct the synthesis of GalK at a level greater than 1% of transcription from P-1. Deletion of the upstream homologies did not affect transcription from P-1. It was concluded that P-1 is responsible for essentially all M1 RNA transcription in vivo. Single-round transcription experiments in vitro detected strong NusA-independent transcriptional pausing at nucleotides +118 and +121 of the rnpB transcript, with a half-life of 27 s when concentrations of NTPs were near the average Km for elongation. Pausing at these points was eliminated by substitution of ITP for GTP in the transcription mixture. This suggests that pausing is dependent on transcript secondary structure. The position of pausing corresponds to that of a dual stem and loop structure of M1 RNA which has recently been proposed on the basis of phylogenetic sequence analysis.

Base Sequence↗
PubMed1989

Teaching human genetics in biochemistry by computer literature searching.

We describe a new user-intense-learning experience that incorporates the teaching of clinical and research applications of human genetics in biochemistry while training first-year medical students to develop skills in computer access to the literature. Human genetics was incorporated into the biochemistry curriculum by providing each student with experience in on-line literature searching in MEDLINE, using Grateful Med, in order to write an abstract about a specific inherited biochemical disorder. We stressed the need for the students to obtain current information in order to understand and interpret the rapidly changing field of human genetics. We taught the students that the most efficient method of obtaining such information was by searching the medical literature via computer.

Biochemistry↗
PubMed1989

[Decoupling or ultra-short therapy of torticollis].

S. Rentrop's survey of literature on the history of the psychotherapeutic treatment of torticollis (76 titles) last year has encouraged us to contribute two examples of treatment to the discussion. The large number of theories, explanations, and treatment methods reveals the wide variety of increasingly effective treatment techniques, but it also makes apparent the dilemma involved in the search for a clear and comprehensive theory. On the basis of two case studies, we would like to show how torticollis symptoms could be eliminated with the help of a one-time focus on a largely unconscious traumatic complex, without additional therapy. One of the two female patients was treated in a supportive clinical environment, the other appeared at the out-patient clinic only once. Finally, we discuss the theoretical approach involved.

Adult↗